Field of the Invention
The invention relates to methods for the highly efficient acquisition of fragment ion mass spectra of precursor ions separated by their mobility in order to identify as many substances as possible in complex substance mixtures, in particular digest peptides in a bottom-up proteomics workflow, or to quantify substances with accurate identity.
Description of the Related Art
In protein science, there is an increasing interest in the identification of as many peptides and proteins as possible in liquid-chromatography/mass-spectrometry runs (LC-MS) of a proteolytic digest of proteins extracted from a biological sample. Mobility spectrometry, coupled to mass spectrometry, has been shown to improve the measurements to reach this goal. As an example for an instrument to perform these measurements, a combination apparatus with an up-front substance separator, e.g. a liquid chromatograph (LC), and a mass spectrometer (MS) may be used, the mass spectrometer equipped with an ion accumulator, an ion mobility separator, a mass filter (usually an RF quadrupole mass filter), an ion fragmentation cell, and downstream a high-resolution time-of-flight mass analyzer with orthogonal ion injection. FIG. 1 gives an example of such a typical instrument. An ion trap may be used to accumulate ions upstream of the ion mobility separator, and a drift tube ion mobility spectrometer to separate the ions by their mobility.
Using this instrument, a mass-mobility map as presented in FIG. 2 is measured, using the mobility separator and the time-of-flight analyzer without filtering masses and fragmenting Ions. The map usually shows the ion masses versus drift time in the drift tube. From this map, an ion species of interest with a specific mobility and mass is selected, indicated by the ellipse in FIG. 2. In a second cycle, fresh ions from the ion accumulator are again separated in time by their ion mobility. The mass filter is adjusted to select the ion species of interest during the specific drift time interval of the ion species, which are then fragmented in the fragmentation cell, and the desired fragment ion spectrum is acquired by the time-of-flight mass analyzer.
In document U.S. Pat. No. 6,960,761 B2 (“INSTRUMENT FOR SEPARATING IONS IN TIME AS FUNCTIONS OF PRESELECTED ION MOBILITY AND ION MASS”, D. E. Clemmer, 2001), this simple data-dependent acquisition method of fragment ions is greatly improved with respect to a better utilization of ions from the ion source. This document describes a large variety of methods to separate ions in mixtures using a number of different combinations of ion sources, ion traps, ion mobility separators, mass filters, collision cells, ion reactors, and high resolution mass analyzers. Furthermore, correspondent apparatuses are described being combined in varying sequence from ion sources, ion traps, ion mobility separators, mass filters, collision cells, ion reactors, and high resolution mass analyzers. Ion traps are used to accumulate ions. Drift tube ion mobility spectrometers with moderate length are applied as ion mobility separators. Among the variations of apparatuses, a device according to FIG. 1 (see Clemmer, FIG. 9 and claims 21 and 22), and an operation method as shown in FIG. 4 can be found (see Clemmer, FIG. 13 and claims 4 and 5). The method with sequential measurements of selected ion species is described in the text (see Clemmer, column 20, line 64 to column 21, line 21). FIG. 3 shows an artificially constructed mass-mobility map of a first measurement cycle. Several (about five following the figures given by Clemmer) ion species of interest are selected in the mass-mobility map. The selected ion species must be sufficiently separated in time by their different mobilities. The time between the selected ion species has to be chosen such that the voltages of the quadrupole mass filter can be switched electronically to filter the selected ion species to be measured next. In a subsequent measurement cycle, fresh ions are accumulated in the ion trap and then separated according to their ion mobility (this part of the method is not clearly outlined by Clemmer and can only be indirectly derived from FIG. 13). If the ion species of interest are not clearly separated by their ion mobilities without any overlap, the ion species are selected in drift time and mass, one after the other, by the mass filter, fragmented in the fragmentation cell, and measured as fragment ion mass spectra by the time-of-flight analyzer. From Clemmer's FIGS. 14 and 15 it can be concluded that about five fragment ion mass spectra can be obtained in a single measuring cycle. In a next cycle, the fragment ion spectra of other ion species selected from the same mass-mobility map may be measured, and so on. In this way, the utilization of ions from the ion source is greatly improved because different ion species being present in a LC peak are separated from each other in time and therefore enriched in their corresponding mobility peaks which are temporally shorter than the LC peak such that more than a hundred fragment ion spectra may be measured in a single second, provided that number of different ion species can be detected in the mass-mobility map. This may not be always the case.
The flow diagram of the essential part of this multi-cycle procedure is outlined in FIG. 4. The diagram describes one of the methods invented by D. E. Clemmer more than a dozen years ago, but introduces new terms “measurement loop” and “measurement cycle” for the sake of clarity.
In document WO 2013/140132 A2 (“MULTI-DIMENSIONAL SURVEY SCANS FOR IMPROVED DATA DEPENDENT ACQUISITIONS” K. Giles and J. L. Wildgoose, 2013), the method is generalized to separations by two different ion characteristics, for example mass and mobility.
At present, there are two commercial instruments on the market which correspond to the type presented in FIG. 1. The Agilent 6560 Ion Mobility Quadrupole Time-of-Flight LC/MS system features a trapping funnel for ion accumulation and an eighty-centimeter drift tube (Rmob≈60) for mobility scans with about fifty milliseconds drift duration for the complete range of mobilities. The Waters Vion IMS QToF mass spectrometer uses a traveling wave ion mobility spectrometer (Rmob≈30) to separate ions by their ion mobility. In both instruments, a prolongation of the scan duration, e.g. by lowering the drift field voltage, decreases the ion mobility resolution. The highest ion mobility resolution will be reached with the highest ion mobility scan speed and thus the lowest ion mobility scan duration, however, there are practical limits to obey. Essentially, there is scarcely any possibility to adapt the separation characteristics to the requirements of the analytical task.
The trapped ion mobility spectrometer (“TIMS”) is an ion mobility spectrometer quite different from both drift tubes at constant electric field (in short: drift tubes) and travelling wave mobility spectrometers. An electric field barrier in a gas flow is used to hold back ions by their ion mobility; a decrease of the field barrier releases ions with increasing ion mobility, resulting in an ion mobility spectrum. The extraordinary and unique characteristic of TIMS is the fact that the ion mobility resolution continually increases with increasing scan duration. The TIMS is described in detail in document U.S. Pat. No. 7,838,826 B1 (M. A. Park, 2008).
There is a need to improve methods for the acquisition of high numbers of fragment ion mass spectra from proteolytic digests of proteins extracted from a biological sample, especially with the goal of finding and identifying as many ion species as possible in the mass-mobility map. Highest detectability for the ion species generated in the ion source of a mass spectrometer requires the collection of as many ions as possible. The collection of more ions, however, should not decrease the number of possible measurements of fragment ion mass spectra per unit of time. Furthermore, there is a need for improved methods to quantify substances in proteolytic digests of proteins extracted from a biological sample.